1. Field of the Invention
The invention relates to the treatment of parthogenic infections through the use of chemotherapeutic agents. More specifically, the invention relates to the treatment of infections by parthogens having resistance to conventional chemotherapeutic agents, such as drug resistant malaria.
2. Summary of the Related Art
Malaria is one of the most widespread of human pathogenic diseases, accounting for high morbidity and mortality, particularly in Southeast Asia, Africa and South America. Partial success in the eradication of this disease has been obtained by control of mosquito populations, institution of vaccination programs and treatment with antimalarial drugs. However, multiple resistance to antimalarial drugs has been largely responsible for a resurgence in the incidence and severity of this disease in recent years. Oaks et al., "Malaria, Obstacles and Opportunities, A report of the committee for the study on malaria prevention and control: status review and alternative strategies", Division of International Health, Institute of Medicine, National Academy Press (1991) discloses up to date information about the disease, its clinical aspects, its etiological agent and vector, as well as current difficulties in controlling the disease and other aspects of the present spread of malaria.
Malaria is just one of a variety of human parasitic infections having increased prevalence worldwide. Webster, in Section X of The Pharmacological Basis of Therapeutics, (Gilman et al., Eds.) Eight Edition, Pargamm Press (1991) discusses several factors responsible for the increase in parasitic infections generally, including population growth and crowding, poor sanitation, inadequate control of parasite vectors, introduction of agricultural water control systems, increased population migration, and development of resistance to agents used for chemotherapy or for control of vectors. In fact, acquired drug resistance has become a major public health problem concerning a variety of infectious pathogens, including bacteria and viruses.
Laboratory techniques for in vitro screening of antimalarial drugs are well known in the art. Such techniques utilize the asexual erythrocytic cycle of Plasmodium falciparum in cultured human red blood cells. Trager and Jensen, Science 193: 673-675 (1976) discloses continuous maintenance of human malarial parasites in vitro. Desjardins et al., Antimicrobial Agents and Chemotherapy 16: 710-718 (1979) discloses a method of quantitative assessment of the in vitro antimalarial activity of drugs, using a semiautomated microdilution technique. Chulay et al., Experimental Parasitology 55: 138-146 (1983) discloses a method of assessing in vitro growth of P. falciparum by measuring incorporation of [.sup.3 H]-hypoxanthine. Lambros and Vanderburg, Journal of Parasitology 65: 418-420 (1979) discloses procedures for the synchronization of the erythrocytic stages of P. falciparum in culture, which allows mechanistic interpretation of the activities of antimalarial drugs.
These in vitro systems have been shown to be predictive of the clinical outcome for a variety of agents in the treatment of human malaria. Bitonti et al., Science 242: 1301-1303 (1988) discloses correct in vitro prediction of reversal of chloroquine resistance in P. falciparum by desipramine. Martin et al., Science 235: 899-901 (1987) discloses correct in vitro prediction of chloroquine resistance in P. falciparum by verapimil.
A variety of antimalarial agents have been developed. These agents act on the asexual erythrocytic stages as schizonticidal agents. Chloroquine, quinine, quinidine, mefloquine and pyrimethamine are weak bases that accumulate to high levels in the acidic food vacuoles of the plasmodial parasite and interfere with a variety of cellular processes of the parasite, as well as with its interaction with its erythrocytic host. These agents can be used in conjunction with sulfonamides, sulfones, or tetracyclines. Specific inhibition of the malarial parasite can be attempted through exploitation of a variety of potential targets. Holder et al., Nature 317: 270-273 (1985) discloses the primary structure of the precursor to the three major surface antigens of the P. falciparum merozoites, the form of the malarial parasite that breaks out of the erythrocyte and invades uninfected erythrocytes. Hadley et al., Ann. Rev. Microbial. 40: 451-477 (1986) discusses the cellular and molecular basis of the invasion of erythrocytes by malaria parasites. Queen et al., Antimicrobial Agents and Chemotherapy 34: 1393-1398 (1990) discusses in vitro susceptibility of P. falciparum to compounds that inhibit nucleotide metabolism, a susceptibility grounded in the exclusive reliance of P. falciparum on a salvage pathway for obtaining purine bases and nucleosides, and upon de novo synthesis of pyrimidines. Ferone et al., Molecular Pharmacology 5: 49-59 (1969) and Hitchings and Burchell, Advances in Enzymology 27: 417-468 (1967) teach that pyrimethamine inhibits protozoal dihydrofolate reductase, and thus de novo pyrimidine biosynthesis, to a much greater extent than it inhibits the mammalian dihydrofolate reductase of the host, thus making pyrimethamine a useful chemotherapeutic against malaria.
Unfortunately, drugs such as pyrimethamine are rendered ineffective by the global emergence of resistant strains. Peterson et al., Proc. Natl. Acad. Sci. USA 85: 9114-9118 (1988) discloses that a point mutation in dihydrofolate reductase-thymidilate synthase confers resistance to pyrimethamine in falciparum malaria. Martin et al., Science 235: 899-901 (1987) teaches that chloroquine resistance in P. falciparum arises from the acquired ability of the parasite to prevent intracellular accumulation of the cytotoxic drug. Multiple drug resistance poses a serious clinical problem for treatment of malaria only with the malarial strain P. falciparum. However, this species accounts for over 85% of the cases of human malaria and for most of the mortality resulting from this disease. Shanzer et al., Proc. Natl. Acad. Sci. USA 88: 6585-6589 (1991) teaches that the resistant parasites maintain their cross-resistance towards a variety of drugs in vitro, as well as in vivo, thus enabling investigators to attempt to identify the biochemical mechanisms underlying drug resistance, and to try to overcome such resistance by innovative chemotherapeutic strategies.
There is, therefore, a need for novel chemotherapeutic approaches for the treatment of drug resistant parasites, such as P. falciparum. Such approaches can be useful also in the treatment of other protozoan infections, including leishmaniasis and trypanosomiasis.
Exogenous administration of synthetic oligonucleotides is an emerging approach for inhibiting a variety of infectious agents. Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75: 280-284 (1978) discloses inhibition of replication and gene expression of Rous Sarcoma Virus (RSV) by exogenous oligonucleotides in tissue cultures of chick embryo fibroblasts, thereby preventing transformation of fibroblasts into sarcoma cells. Stephenson and Zamecnik, Proc. Natl. Acad. Sci. USA 75: 285-288 (1978) teaches that the same oligonucleotide inhibits cell-free synthesis of proteins specified by the RSV 305 RNA in a reticulocyte system. Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 (1986) discloses inhibition of replication of human immunodeficiency virus (HIV) in vitro screening systems, using synthetic oligonucleotides that are complementary to a variety of conserved regions of the HIV genome. The use of modified internucleotide bridging phosphates resulted in a 10 to 100-fold decrease in the 50% inhibitory concentration (IC.sub.50) for in vitro HIV replication. Matsukura et al., Proc. Natl. Acad. Sci. USA 84: 7706-7710 (1987) discloses this effect using oligonucleotide phosphorothioates. Agrawal et al., Proc. Natl. Acad. Sci. USA 85: 7079-7084 (1988) shows a similar effect for oligonucleotide phosphorothioates and phosphoroamidates. Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448-7451 (1988) discloses enhanced inhibition of HIV, using oligonucleotide methylphosphonates.
The use of exogenous oligonucleotides to inhibit retroviral infection, as disclosed in the above publications and in Goodchild et al., U.S. Pat. No. 4,806,463, represents treatment of a latent or dormant condition, since the retroviral genome is integrated into the host cell genome and is expressed with the participation of host cellular enzymes and factors only after a significant latency period. In contrast, the treatment of malaria, other infectious parasitic diseases and acute viral and bacterial infections represents chemotherapy for active infections requiring immediate treatment. Bzik, et al., Proc. Natl. Acad. Sci. USA 84: 8360-8364 (1987) teaches the nucleotide sequence of the P. falciparum dihydrofolate reductase-thymidilate synthese gene. However, recent attempts at using exogenous oligonucleotides to inhibit synthesis of these proteins from P. falciparum mRNA in a cell free translation system have shown an absence of promise for this approach for the clinical treatment of malaria. Sartorius and Franklin, Nucleic Acids Res. 19: 1613-1618 (1991) demonstrates a complete failure of oligonucleotides to inhibit protein synthesis in such a system, unless the oligonucleotides are pro-annealed to P. falciparum mRNA at an elevated temperature of 65.degree. C. for 5 minutes, followed by a one hour cooling at 30.degree. C. Moreover, even under these highly nonphysiological conditions a dramatically high concentration of 150-170 .mu.M was required for the 30-49 nucleotide oligomers to produce 50% inhibition. These results suggest that inhibition of malarial protein synthesis by oligonucleotides will not be possible in vivo, where the host erythrocyte and the intraerythrocytic parasite are maintained at the body temperature of 37.degree. C.